A new generation of nanobody research tools using improved mass spectrometry-based discovery methods

Single-domain antibodies (“nanobodies”) derived from the variable region of camelid heavy-chain only antibody variants have proven to be widely useful tools for research, therapeutic, and diagnostic applications. In addition to traditional display techniques, methods to generate nanobodies using direct detection by mass spectrometry and DNA sequencing have been highly effective. However, certain technical challenges have limited widespread application. We have optimized a new pipeline for this approach that greatly improves screening sensitivity, depth of antibody coverage, antigen compatibility, and overall hit rate and affinity. We have applied this improved methodology to generate significantly higher affinity nanobody repertoires against widely used targets in biological research—i.e., GFP, tdTomato, GST, and mouse, rabbit, and goat immunoglobulin G. We have characterized these reagents in affinity isolations and tissue immunofluorescence microscopy, identifying those that are optimal for these particularly demanding applications, and engineering dimeric constructs for ultra-high affinity. This study thus provides new nanobody tools directly applicable to a wide variety of research problems, and improved techniques enabling future nanobody development against diverse targets.

CALL001/CALL002 PCR amplicons from llama or alpaca lymphocyte cDNA were sequenced by PacBio, and the most abundant VHH N-term and C-term 18 bp sequences were identified to assess potential priming locations.Percentage of the total analyzed population is indicated for each 18bp sequence.(C) Hinge PCR primers were searched against lymphocyte mRNA sequences to estimate relative coverage.The top 10 complementary sequence hits are shown, with percentage of total matches.Exact primer matches are indicated in blue, and mismatched nucleotides labeled in red.SH-rev matches also included off-target non-IgG sequences, indicated by asterisks.

Fig S2 .
Fig S2.Comparing VHH coverage of nested PCR and single-step hinge PCR.(A) Schematic comparison of traditional nested PCR and single-step hinge PCR protocols.(B) CALL001/CALL002 PCR amplicons from llama or alpaca lymphocyte cDNA were sequenced by PacBio, and the most abundant VHH N-term and C-term 18 bp sequences were identified to assess potential priming locations.Percentage of the total analyzed population is indicated for each 18bp sequence.(C) Hinge PCR primers were searched against lymphocyte mRNA sequences to estimate relative coverage.The top 10 complementary sequence hits are shown, with percentage of total matches.Exact primer matches are indicated in blue, and mismatched nucleotides labeled in red.SH-rev matches also included off-target non-IgG sequences, indicated by asterisks.

Figure S7 .
Figure S7.Screening GFP nanobodies by immunofluorescence.(A) Schematic of nanobody conjugation and staining procedure.Nanobodies were labeled with Alexa Fluor 568 and used to stain brain sections of Thy1-GFPM mice.(B) Fluorescent imaging was performed on GFP or nanobody-AF568 signal.(C) Quantitative comparisons of relative GFP and nanobody fluorescence.Fluorescence was normalized to maximum fluorescence in each channel.Plots were fitted with simple linear regression, and Pearson correlation coefficients (r) are indicated.Scale bars are 100 µm.Micrographs and quantification panels for LaG94-12, LaG94-15, and LaG94-18 are reproduced here from Fig. 4C to aid in comparison between samples.

Figure S8 .
Figure S8.Screening tdTomato nanobodies by immunofluorescence.(A) Schematic of nanobody conjugation and staining procedure.Nanobodies were labeled with Alexa Fluor 647 and used to stain sagittal brain sections of ChAT-Cre::Ai14 mice, with tdTomato expressed in cholinergic neurons.(B) Fluorescent imaging was performed on tdTomato or nanobody-AF647 signal.(C) Quantitative comparison of relative tdTomato and nanobody fluorescence.Fluorescence was normalized to maximum fluorescence in each channel.Plots were fitted with simple linear regression, and Pearson correlation coefficients (r) are indicated.Scale bars are 100 µm.Micrographs and quantification panels for LaTdT-1 and LaTdT-39 are reproduced here from Fig. 4D to aid in comparison between samples.

Figure S13 .
Figure S13.Screening GFP nanobody dimers by immunofluorescence.GFP nanobody dimers were labeled with Alexa Fluor 647 and used to stain brain sections of Thy1-GFPM mice.(A) Fluorescent imaging was performed on GFP or nanobody-AF647 signal.(B) Quantitative comparisons of relative GFP and nanobody fluorescence.Fluorescence was normalized to maximum fluorescence in each channel.Plots were fitted with simple linear regression, and Pearson correlation coefficients (r) are indicated.Scale bars are 100 µm.Micrographs and quantification panels for LaG 94-1--94-14 are reproduced here from Fig. 4E to aid in comparison between samples.

Figure S14 .
Figure S14.Screening IgG nanobodies by immunofluorescence.(A) Schematic of nanobody conjugation and staining procedure.IgG nanobodies were labeled with Alexa Fluor 568 or 647 and used to stain brain sections of Thy1-GFPM probed with an anti-GFP antibody of the corresponding species (mouse or rabbit).For goat IgG nanobodies, somatostatin tdTomato mouse brain sections were stained with goat anti-tdTomato.Fluorescent imaging was performed on GFP/tdTomato or nanobody signal using (B) mouse IgG nanobodies, (C) rabbit IgG nanobodies, or (D) goat IgG nanobodies.Traditional rabbit or goat secondaries were also used as controls.Quantitative comparisons of relative GFP or tdTomato and nanobody/secondary fluorescence are plotted at right.Fluorescence was normalized to maximum fluorescence in each channel.Plots were fitted with simple linear regression, and Pearson correlation coefficients (r) are indicated.Scale bars are 100 µm.Micrographs and quantification panels for LaMIgG-8, LaMIgG-14, and Goat Anti Mouse are reproduced from Fig. 5A in (B); LaRIgG-1 and LaRIgG-2 from Fig. 5B in (C); and LaGIgG-12 and Donkey Anti Goat from Fig. 5C in (D), to aid in comparison between samples.

Figure S15 .
Figure S15.Species and isotype specificity of IgG nanobodies.Nanobodies were labeled with Alexa Fluor 488 and used to probe immobilized IgG of the indicated species, fragment, and/or isotype.An anti-GFP nanobody (LaG) was used as a negative control.